Hajime Hada

Loss of heterozygosity and analysis of mutation of p53 in hepatocellular carcinoma.

Abstract Thirty‐six hepatocellular carcinoma (HCC) tissues obtained from 34 patients were classified according to histological diagnosis into six well‐differentiated HCC, 20 moderately differentiated HCC and 10 poorly differentiated HCC. High molecular weight DNA was prepared from each tumour and the corresponding non‐tumour tissue. Loss of heterozygosity (LOH) on chromosomes 4q, 5q, 10q, 11p, 16q, 17p, mutation of the p53 gene and polymorphism of intron 25 of the retinoblastoma (RB) gene were simultaneously analysed. The patients were composed of three cases of small HCC (the diameter of which was < 3 cm) and 31 cases of advanced HCC. Twenty‐nine of 34 (85.3%) patients analysed had been exposed to hepatitis B virus and/or hepatitis C virus. The frequencies of LOH on seven chromosomes were 57.9% in 17p13.3, 45.1% in 17p, 45.1% in 11p, 41.9% in 5q, 41.9% in 16q24, 29.0% in 4q, 25.8% in 10q in advanced HCC (four of well differentiated, 18 of moderately differentiated and nine of poorly differentiated carcinoma). In contrast, LOH was observed on 4q, 5q, 16q and 17p in 33% (1/3) of the small HCC (two of well differentiated and one of moderately differentiated carcinoma).

Improvement of serum amino acid profile in hepatic failure with the bioartificial liver using multicellular hepatocyte spheroids

We designed a bioartificial liver support system in which encapsulated multicellular spheroids of rat hepatocytes were utilized as a bioreactor in a hollow fiber cartridge. The spheroids, formed in a positively charged polystyrene dish that contained hormonally defined medium, were encapsulated into microdroplets of agarose that contained about 9 × 107 rat hepatocytes. The medium, including 150 mL reservoir volume, was circulated in a closed circuit in which the cartridge was inserted. The pH and levels of dissolved oxygen were monitored and automatically regulated so that they were maintained within a constant range for 72 h. Albumin accumulated in the circuit at the rate of 2.0 mg/L/h in this system. When the bioreactor cells in the system were replaced with Hep G2 cells, a human hepatoblastoma cell line, albumin accumulated at the rate of 0.15 mg/L/h. The spheroids of primary culture hepatocytes had 13 times higher albumin‐producing capacity than the aggregates of Hep G2. The serum of a patient with fulminant hepatic failure was circulated in this system with the spheroids of primary culture hepatocytes. The concentration of branched amino acid (BCAA) in the circuit significantly increased during the 48 h circulation, while the concentration of aromatic amino acid (AAA) and methionine decreased. The ratio of BCAA/AAA increased from 0.640 to 0.772, indicating that the hepatocyte spheroids had improved the imbalance of the amino acid profile in the serum. These findings indicate that this system may be a useful model for an artificial liver support.

Treatment of hepatocellular carcinoma by transcatheter hepatic arterial injection of radioactive iodized oil solution

SummaryAfter 12 days of culture, VX2 carcinoma cells were inoculated into the liver of 16 rabbits; 14 days later,131I-labeled iodized oil ([131I]-Lp) suspended in lipiodol was injected into the hepatic artery. Selective accumulation of the contrast material in the tumor for an extended time was evident on X-rays and hepatic scintiphotographs. The antitumor effect was remarkable. [131I]-Lp agents warrant further examination for their clinical usefulness. Internal radiation therapy by transcatheter hepatic arterial injection of [131I]-Lp (group A) was evaluated in 9 patients with hepatocellular carcinoma (HCC, tumor stage III or IV) associated with liver cirrhosis (LC) and compared with combination therapy of Lp-TAE (group B) in 18 patients with HCC (tumor stage III or IV) associated with LC. In group A, serum AFP levels dropped rapidly in eight of the nine patients who had an elevated intial level of more than 500 ng/ml. The average reduction in tumor size was 50% in eight cases as determined by computed tomography. Histological examination of one resected liver specimen at 3 months after the third injection of [131I]-Lp revealed microscopic features highly suggestive of a radiation effect in the [131I]-Lp-containing area. The 1-year survival value for patients with HCC was estimated at 49.0% using the Kaplan-Meier method. The survival of patients treated with internal radiation therapy tended to be better than that of those treated with Lp-TAE (P=0.119).

Molecular hybridization methods for determination of serum HBV-DNA

SummaryMolecular hybridization methods for determination of hepatitis B virus DNA (HBV-DNA) in serum were studied. A simple method by which serum was treated with sodium hydroxide, followed by dot hybridization procedure on filter sheets provides a sensitive and direct result for detecting HBV-DNA. Another method in which DNAs extracted from Dane particle fraction were subjected to the molecular hybridization method on a filter membrane, provided similar results although this method is time consuming. The third method in which serum was directly spotted on filter sheets, followed by alkalinetreatment seems to be less sensitive. Three filter papers, NC filter, Zeta-Probe and Biodyne, on which molecular hybridization was performed, gave similar sensitivity.

Hepatitis B virus dna in human hepatocellular carcinoma: is the integration of hepatitis B virus dna really carcinogenic?

SummaryHepatitis B virus-specific DNA sequences (HBVDNA) in the liver were examined in 19 patients with hepatocellular carcinoma (HCC), 5 patients with liver cirrhosis (LC) and without HCC, 3 patients with chronic hepatitis (CH), 2 patients with metastatic liver cancer (MLC), 1 patient with primary biliary cirrhosis (PBC) and 4 patients with normal liver (NL) by the Southern blot hybridization procedure. Integration of HBVDNA was found in all 4 HCC patients with serum HBsAg, of whom one patient had HBVDNA only in the non-tumor (cirrhotic) region. Integration of HBVDNA was also detected in 4 of 8 HCC patients without serum HBsAg but with serum HBV-related antibodies, and in 2 HBsAg-positive patients of 5 LC patients. All 3 HBsAg-positive CH patients had only extrachromosomal HBVDNA. No HBVDNA was detected in the other 21 patients. Although integration of HBVDNA was observed in HBsAg-positive HCC patients with a higher frequency, integrated HBVDNA could also be detected in non-tumor regions of HCC patients and cirrhotic livers without HCC. It was concluded from these observations that integration of HBVDNA was frequently associated with HCC but might not have a direct causal effect on hepatocarcinogenesis even in HBV carriers.

Classification of hepatitis C virus into subgroups on the basis of sequence variations in the envelope protein

In order to investigate the possibility that hepatitis C virus (HCV) can be classified into subgroups based on the variations in the envelope region, the nucleotide and deduced amino acid sequences were compared among the reported viral isolates including HCV1, HCJ1, HCJ4, HCVJ, HCVBK, HCVNK and HCJ6. From the homology analysis, the HCV isolates were classified into three subgroups: group I (HCV1 and HCJ1); group II (HCJ4, HCVJ, HCVBK and HCVNK); and group III (HCJ6). Furthermore, two novel regions were found in the E1 envelope region. One is located at aa246–258 (intersubtype variable region 1; ISVR‐1) where the amino acid sequences were relatively conserved within each subgroup, while the sequences were extremely different among the subgroups. Another is located at aa315–328 (intersubgroup common region‐1; ISCR‐1) where the amino acid sequences were completely identical among all seven HCV isolates despite the fact that the marked variations were distributed throughout the envelope region. The results suggest that the combination of ISVR‐1 and ISCR‐1 can be utilized as marker sequences for the classification of HCV strains and that the letter region might be one of the candidates for the vaccine.